Multi-repeat sequences identification using genome mining techniques for developing highly sensitive molecular diagnostic assay for the detection of Chlamydia trachomatis

Abstract

Chlamydia trachomatis ( C. trachomatis) is a common sexually transmitted infection (STI). In 2019, the World Health Organization reported about 131 million infections. The majority of infected patients are asymptomatic with cases remaining undetected. It is likely that missed C. trachomatis infections contribute to preventable adverse health outcomes in women and children. Consequently, there is an urgent need of developing efficient diagnostic methods. In this study, genome-mining approaches to identify identical multi-repeat sequences (IMRS) distributed throughout the C. trachomatis genome were used to design a primer pair that would target regions in the genome. Genomic DNA was 10-fold serially diluted (100pg/μL to 1×10 ^-3pg/μL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and products were resolved on agarose gel. The novel assay, C. trachomatis IMRS-PCR, had an analytical sensitivity of 4.31 pg/µL, representing better sensitivity compared with 16S rRNA PCR (9.5 fg/µL). Our experimental data demonstrate the successful development of lateral flow and isothermal assays for detecting C. trachomatis DNA with potential use in field settings. There is a potential to implement this concept in miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for reliable point-of-care testing.

Keywords: Chlamydia trachomatis; Identical Multi-Repeat Sequences; Isothermal assays.

Figure 1.. Chlamydia trachomatis-IMRS primer targets and gel images from the C. trachomatis IMRS and conventional 16S rRNA PCR assay.

( A) Circos plot for the distribution of identical multi repeat sequence (IMRS) primers in the C. trachomatis genome. C. trachomatis IMRS primer A (blue lines) IMRS primer B (red lines) both have 6 repeats. ( B) and ( C) Gel image of 10-fold serially diluted (1 – 100, 2 – 10, 3 – 1, 4 – 0.1, 5 – 0.01, 6 – 0.01, 7 – 0.001 and 8 - NTC) (pg/μl) genomic C. trachomatis DNA amplicons resolved on 1% gel using IMRS primers and gold standard 16S rRNA PCR primers, respectively.

Figure 2.

Real-time PCR amplification plots from the Chlamydia trachomatis-identical multi repeat sequence (IMRS) ( A) and the 16S rRNA ( B) assays using serially diluted C. trachomatis genomic DNA.

Figure 3.. Probit regression analysis to estimate the lower limit of detection for the Chlamydia trachomatis-identical multi repeat sequence (IMRS) primers and the 16S rRNA PCR.

Probit analysis estimation for C. trachomatis-IMRS ( A). As indicated, the IMRS primers for C. trachomatis had an LLOD = 9.5 fg/μl. B: Probit analysis estimation for 16S rRNA PCR. As indicated, gold standard primers for C. trachomatis had an LLOD = 4.31 pg/μl.

Figure 4.. Chlamydia trachomatis-Iso-identical multi repeat sequence (IMRS), lateral flow assay and estimation of the lower limit of detection of the isothermal assay.

( A) Gel image of C. trachomatis-Iso IMRS assay products visualized on 1% gel. Five replicates of each dilution served as DNA template for the C. trachomatis-Iso IMRS. DNA concentration is in genome copies per μl. ( B) Visual read-out detection of serially diluted C. trachomatis DNA using the lateral flow assay. Amplicons were incubated at 65°C for 1 hour and transferred onto strips as indicated. IC – Internal Control, NTC – Non Template Control. ( C) Probit analysis estimation for C. trachomatis Iso-IMRS. As indicated, the IMRS primers for C. trachomatis had an LL0D = 0.3162 ng/μl.

Figure 5.. Transformed E.coli cells expressing Chlamydia trachomatis sequences amplified using C. trachomatis-identical multi repeat sequence (IMRS) primers.

Figure 6.. Mean Ct values of PCR confirmed 16 clinical DNA samples from RT-PCR assay that were used to validate the Chlamydia trachomatis-identical multi repeat sequence (IMRS) PCR primers.

Figure 7.. Gel image of PCR products obtained after amplification of Treponema pallidum (TP) and Trichomonas vaginalis (TV) genomic DNA using Chlamydia trachomatis-identical multi repeat sequence (IMRS) primers.

C. trachomatis genomic DNA was used as a positive control. N = Negative control, L = Ladder.